Dataset Information

Lentiviral delivery of PPAR? shRNA alters the balance of osteogenesis and adipogenesis, improving bone microarchitecture.

ABSTRACT:

Introduction

Skeletal aging is associated not only with alterations in osteoblast (OB) and osteoclast (OC) number and activity within the basic metabolic unit, but also with increased marrow adiposity. Peroxisome proliferator-activated receptor gamma (PPAR?) is commonly considered the master transcriptional regulator of adipogenesis, however, it has known roles in osteoblast and osteoclast function as well. Here, we designed a lentiviral delivery system for PPAR? shRNA, and examined its effects in vitro on bone marrow stromal cells (BMSC) and in a mouse intramedullary injection model.

Methods

PPAR? shRNA was delivered by a replication-deficient lentiviral vector, after in vitro testing to confirm purity, concentration, and efficacy for Pparg transcript reduction. Next, control green fluorescent protein lentivirus or PPAR? shRNA expressing lentivirus were delivered by intramedullary injection into the femoral bone marrow of male SCID mice. Analyses included daily monitoring of animal health, and postmortem analysis at 4 weeks. Postmortem analyses included high resolution microcomputed tomography (microCT) reconstructions and analysis, routine histology and histomorphometric analysis, quantitative real time polymerase chain reaction analysis of Pparg transcript levels, and immunohistochemical analysis for markers of adipocytes (PPAR?, fatty acid binding protein 4 [FABP4]), osteoblasts (alkaline phosphatase [ALP], osteocalcin [OCN]), and osteoclasts (tartrate-resistant acid phosphatase [TRAP], Cathepsin K).

Results

In vitro, PPAR? shRNA delivery significantly reduced Pparg expression in mouse BMSC, accompanied by a significant reduction in lipid droplet accumulation. In vivo, a near total reduction in mature marrow adipocytes was observed at 4 weeks postinjection. This was accompanied by significant reductions in adipocyte-specific markers. Parameters of trabecular bone were significantly increased by both microCT and histomorphometric analysis. By immunohistochemical staining and semi-quantification, a significant increase in OCN+osteoblasts and decrease in TRAP+multinucleated osteoclasts was observed with PPAR? shRNA treatment.

Discussion

These findings suggest that acute loss of PPAR? in the bone marrow compartment has a significant role beyond anti-adipose effects. Specifically, we found pro-osteoblastogenic, anti-osteoclastic effects after PPAR? shRNA treatment, resulting in improved trabecular bone architecture. Future studies will examine the isolated and direct effects of PPAR? shRNA on OB and OC cell types, and it may help determine whether PPAR? antagonists are potential therapeutic agents for osteoporotic bone loss.

SUBMITTER: James AW

PROVIDER: S-EPMC4195482 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Lentiviral delivery of PPARγ shRNA alters the balance of osteogenesis and adipogenesis, improving bone microarchitecture.

James Aaron W AW Shen Jia J Khadarian Kevork K Pang Shen S Chung Greg G Goyal Raghav R Asatrian Greg G Velasco Omar O Kim Jung J Zhang Xinli X Ting Kang K Soo Chia C

Tissue engineering. Part A 20140729 19-20

<h4>Introduction</h4>Skeletal aging is associated not only with alterations in osteoblast (OB) and osteoclast (OC) number and activity within the basic metabolic unit, but also with increased marrow adiposity. Peroxisome proliferator-activated receptor gamma (PPARγ) is commonly considered the master transcriptional regulator of adipogenesis, however, it has known roles in osteoblast and osteoclast function as well. Here, we designed a lentiviral delivery system for PPARγ shRNA, and examined its ...[more]

PMID: 24785569

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